Neurofilaments are major, but nonetheless poorly understood, neuron specific 10nm filaments. They are composed of three subunit proteins usually referred to as NF-1, NF-M and NF-H. NF-M and NF-H differ from other characterized 10 nm filament proteins in that they contain long heavily phosphorylated carboxyterminal extensions. These carboxyterminal regions have recently been shown to contain two distinct segments, probably corresponding to two types of functional domain. The first contains multiple repeats of 5-8 amino acid segments each containing the sequence lysine-serine-proline, and is here referred to as KSP segment. The second domain, at the extreme carboxyterminus, has a high content of lysine and glutamic acid, and is called the KE segment. In the present proposal I wish to test two hypotheses I have proposed which assign functions to these unusual segments. These hypotheses are; 1. That some or all of the KE segments are responsible for a strong interaction which binds neurofilaments together into bundles. i will search for such an interaction using isolated KE domains and fusion proteins containing KE segments. Detection of a binding interaction will be followed by characterization of his interaction in detail and proof that this interaction is actually responsible for filament bundling in vivo. 2. That phosphorylation of the KSP regions of the NF-H and NF-M molecules is responsible for modulating the spacing of neurofilaments from one another in bundles. Evidence for or against this hypothesis will not detract from the importance of identifying and characterizing the KSP kinase activity, which may phosphorylate other axonal proteins besides neurofilaments and appears to change in activity following neuronal injury and in various disease states. I will make use of the new sequence data to isolate this enzyme. In summary the studies proposed will increase our understanding of the function of two unusual regions of the neurofilament molecules, and will characterize a kinase activity of great potential importance in recovery from injury and in the maintenance of differences between axons and dendrites.